Charm Sciences, Inc. ROSA DONQ2 Quantitative Test

1 United States Department of Agriculture Test Kit Instructions: LF- DONQ2 Revision 1 Grain Inspection, Packers and Stockyards Administration Effective Date: 01/07/2015 Technology and Science Division Charm Sciences, Inc. rosa DONQ2 Quantitative Test Section Page Number GENERAL INFORMATION .. 1 PREPARATION OF TESTING MATERIALS AND EQUIPMENT .. 2 SAMPLE PREPARATION AND EXTRACTION PROCEDURES .. 3 TEST PROCEDURES .. 4 SUPPLEMENTAL ANALYSIS .. 6 REPORTING AND CERTIFYING TEST RESULTS .. 7 STORAGE CONDITIONS AND PRECAUTIONS .. 7 EQUIPMENT AND SUPPLIES .. 8 REVISION HISTORY .. 9 United States Department of Agriculture Test Kit Instructions: LF- DONQ2 Revision 1 Grain Inspection, Packers and Stockyards Administration Effective Date: 01/07/2015 Technology and Science Division P a g e | 1 GENERAL INFORMATION rosa DONQ2 Quantitative Test is an immunoreceptor assay utilizing rosa (Rapid One Step Assay) lateral flow technology.

2 DON (deoxynivalenol or vomitoxin) is extracted from the samples using water. DON interacts with colored beads in the lateral flow test strip and the color intensity in the test and control zones is measured by the rosa -M Reader or Charm EZ-M reader and interpreted as parts per million (ppm) DON. The instructions presented in this document cover only the procedure for performing the analytical test for official inspections. For questions regarding this procedure, contact Dr. Ajit Ghosh of the Technology and Science Division by phone at 816-891-0417 or email at Refer to the current policies and/or instructions issued by the Policies, Procedures, and Market Analysis Branch (PPMAB) of the Field Management Division for information on use of this test kit in official inspections including sampling, general sample preparation ( , grinding and dividing), reporting and certification of test results, laboratory safety, and hazardous waste management.

3 For questions regarding these policies and/or instructions, contact Patrick McCluskey of PPMAB by phone at 816-659-8403 or email at Approved Test Kit Information Test Kit Vendor: Charm Sciences, Inc. 978-687-9200 Test Kit Name: rosa DONQ2 Quantitative Test Product Number: LF- DONQ2 Effective Date of Instructions: 01/07/2015 Instructions Revision Number 1 Conformance Range: ppm Number of Analyses to Cover Conformance Range: 1 Type of Service: Quantitative Supplemental Analysis: Yes Approved Commodities: Corn, wheat, barley, brewer s rice, buckwheat, corn bran, corn germ meal, corn gluten feed, corn gluten meal, distillers dried grain with solubles, hominy, malted barley, milled rice, oats, rapeseed meal, rice bran, rough rice, rye, sorghum, soybean meal, triticale, wheat bran, wheat flour, wheat middlings, wheat red dog, wheat screenings Extraction method.

4 Shake vigorously 50 grams ground sample with 250 milliliters (mL) of deionized or distilled water for 1 minute. Test Format: Lateral flow strip Detection Method: rosa -M Reader, Model LF-ROSAREADER-M-NB Charm EZ-M, Model LF- rosa -EZ-M United States Department of Agriculture Test Kit Instructions: LF- DONQ2 Revision 1 Grain Inspection, Packers and Stockyards Administration Effective Date: 01/07/2015 Technology and Science Division P a g e | 2 PREPARATION OF TESTING MATERIALS AND EQUIPMENT a. Test Strips: Remove from the container only the number of test strips to be used in 1 day, document time of removal. Keep these test strips at room temperature during daily use for up to 12 hours and unused test strips should be discarded.

5 B. DONQ2 Dilution Buffer: (1) Dispense buffer into a clean micro-centrifuge tube and label for each sample to be tested. (2) Use pre-dispensed buffer tubes and buffer solution at room temperature (18 to 30 C). c. Negative Control: DONQ2 Dilution Buffer is used as a negative control. d. Positive Control: (a) Reconstitute the dry positive control (provided with test kit) by adding mL DONQ2 Dilution Buffer. Shake well; allow to stand for 10 minutes at room temperature before use, and mix again just before use. (b) Store reconstituted positive control refrigerated (0 to 7 C) for up to 1 week or aliquot (at least mL) to clean micro-centrifuge tubes, label, and freeze within 6 hours of reconstitution (-15 C or below) for up to 2 months.

6 Thaw slowly (overnight in refrigerator or with cool water) and shake well before use. Store thawed positive control refrigerated and use within 24 hours of thawing; DO NOT REFREEZE. e. Reader and Test Strip Performance Testing: (1) Equipment Setup (a) rosa -M Reader: Enter performance mode in rosa -M Reader by selecting DON channel in 3-line mode (DON flashing) and sequentially pressing ESC, 5, ENTER. Follow rosa -M Reader prompts to test calibration strips (LOWCAL and HIGHCAL) and controls (NEGCONTROL and POSCONTROL). (b) Charm EZ-M reader: Enter performance mode in Charm EZ-M by selecting Perf. Mon. from the Main Menu, followed by Perf. Test. Follow Charm EZ-M prompts to test calibration strips (LO CAL and HI CAL) and controls (NEG CTRL and POS CTRL).

7 Select DONQ2 from the TESTS list if prompted. (2) Test calibration strips daily to verify rosa -M Reader or Charm EZ-M performance. Calibration strips must test/perform in the specified ranges. (3) Test negative control and positive control weekly to verify test strip performance. Valid control ranges are: (a) Negative Control: less than or equal to 100 ppb ( ppm) (b) Positive Control: 500 to 1500 ppb ( to ppm) If calibration strips or controls do not perform in specified ranges, discontinue use and contact Charm Sciences for assistance. Notify your monitoring field office or TSD with any documented information for quality control purposes. United States Department of Agriculture Test Kit Instructions: LF- DONQ2 Revision 1 Grain Inspection, Packers and Stockyards Administration Effective Date: 01/07/2015 Technology and Science Division P a g e | 3 f.

8 rosa Incubator: rosa Incubator must be clean and level. The rosa Incubator temperature must be at 45 1 C (the temperature indicator should match the incubator temperature). SAMPLE PREPARATION AND EXTRACTION PROCEDURES a. Procedure for barley, brewer s rice, buckwheat, corn, corn bran, corn gluten feed, corn germ meal, corn gluten meal, hominy, malted barley, milled rice, oats, rapeseed meal, rough rice, rye, sorghum, soybean meal, triticale, wheat, wheat bran, wheat flour, wheat red dog, wheat screenings: (1) Weigh 50 grams ground samples into a clean extraction container. (2) Add 250 mL deionized or distilled water. (3) Shake vigorously for 1 minute. (4) Allow sample to settle for 1 minute to obtain settled extract (use within 30 minutes).

9 (5) Transfer 1 to mL settled extract into a clean micro-centrifuge tube, label, and centrifuge for 10 seconds (use within 2 hours). (6) Pipet mL DONQ2 Dilution Buffer into a clean micro-centrifuge tube. (7) Pipet 50 microliters ( L) centrifuged sample extract to micro-centrifuge tube containing mL DONQ2 Dilution Buffer, cap, mix (5 times inverting up and down), and label. This sample is the Diluted Extract, and ready for the test (use within 6 hours after preparation). (8) Repeat steps 1 to 7 for additional samples. b. Procedure for distillers dried grains with solubles: (1) Weigh 50 grams ground samples into a clean extraction container. (2) Add 250 mL deionized or distilled water. (3) Shake vigorously for 1 minute.

10 (4) Allow sample to settle for 1 minute to obtain settled extract (use within 30 minutes). (5) Transfer 10 to 15 mL settled extract into a clean conical tube and label. (6) Adjust pH of settled extract by adding 10-30% KOH drop wise until pH is to Monitor pH with pH strips or pH meter. (7) Transfer 1 to mL pH adjusted extract into a clean micro-centrifuge tube, label, and centrifuge for 10 seconds (use within 2 hours). (8) Pipet mL DONQ2 Dilution Buffer into a clean micro-centrifuge tube. (9) Pipet 50 L centrifuged sample extract to micro-centrifuge tube containing mL DONQ2 Dilution Buffer, cap, mix (5 times inverting up and down), and label. This sample is the Diluted Extract, and ready for the test (use within 6 hours after preparation).